Although most patients with Acute Promyelocytic Leukemia (APL, FAB M3 AML) have good outcomes, 15% still die from relapsed disease. The long-term goal of this work is to understand the genetic events that contribute to the pathogenesis of APL, so that better therapies can be developed for patients who relapse. We and others have established that PML-RARA can initiate APL in mice, yielding an ATRA-, arsenic-, and chemo-responsive APL-like disease that has long latency and inappropriate myeloid differentiation, suggesting that this model is still missing important genetic elements that 'shape'the myeloid maturation arrest observed in APL patients. To better understand the key genetic events controlling APL initiation and progression, we propose the following Specific Aims: Specific Aim 1: We will define the role of RARA haploinsufficiency for APL pathogenesis. RARA haploinsufficiency is a consequence of t(15;17), but its role has not yet been defined for APL pathogenesis. We have developed a new mouse model of APL by targeting a conditional PML-RARA bcr1 fusion cDNA to the 5'untranslated region of the mouse PML gene in B6 mice, creating haploinsufficiency for PML, and appropriate expression levels of PML-RARA. By crossing these mice with RARA haploinsufficient mice, we have created animals with three of the four potentially relevant somatic events caused by t(15;17). The APL phenotype of these animals (with and without RARA haploinsufficiency) will be fully defined. Specific Aim 2: We will identify the genomic DNA binding sites of PML-RARA and the genes that it directly regulates. We have developed a functional 3x-FLAG-tagged PML-RARA protein (FLAG-PR) that is capable of interacting with RXRA, and binding to DNA sequences that contain known RARA binding sites. FLAG-PR will be used as the 'bait'for chromatin immunoprecipitation of DNA;PML-RARA binding sites in the chromatin of PR-9 cells and APL cells will be resolved by Chip-Seq and parallel ChIP-on-chip studies. Expression data from APL patients will be used to identify possible genes that are directly regulated by PML- RARA, and candidate genes will be validated. Specific Aim 3: We will define progression mutations that contribute to APL pathogenesis. Using the Illumina platform, we will sequence the entire genomes of 15 APL tumors derived from C57Bl/6 mice that express PML-RARA under control of the murine cathepsin G locus, and ultimately, the novel mice described in Aim 1. Since these tumors arose in inbred mice, only 10x coverage of each genome (i.e. ~27 billion base pairs of sequence) should be required to discover all acquired mutations. The frequency of validated mutations will assessed in both mouse and human APL/AML samples, and recurrent mutations will be studied for functional consequences in vitro and in vivo. PUBLIC HEALTH RELEVANCE: Acute promyelocytic leukemia (APL/M3 AML) is a subtype of acute myeloid leukemia (AML) that has a well- defined genetic initiating event and effective treatment options, but ~15% of patients with APL die from progressive disease despite the best current therapies. This grant will help us to define all of the genetic events that contribute to the initiation and progression of this disease, which should allow us to develop better strategies for treating relapsed APL patients. These studies may also help us understand other AML subtypes, since these patients often have progression mutations that are also found in APL genomes.